The long term goal of this research is to elucidate the structural organization and regulatory mechanisms involved in testes-specific expression of the human Ldh-c gene which encodes lactate dehydrogenase-C4 (LDH-C4: also referred to as LDH-X). Human genomic libraries will be screened with a cDNA probe comprising the complete region of the human LDH-C subunit as well as part of the 5' nontranslated region and the complete 3' nontranslated region. Genomic clones will be isolated and characterized for exon-intron boundaries. 3' and 5' fragments will be subcloned into M13mp19 for sequencing by the Sanger dideoxy procedure using the Klenow fragment of DNA polymerase and 35SdATP as a tracer. To produce large quantities of catalytically active LDH-C- for biochemical characterization and antibody production, the Ldh-c cDNA will be subcloned into expression vectors (E.coli and /or Baculovirus) after modifying the 5' and 3' ends by recombinant DNA methodologies. The chromatin structure of the Ldh-c gene in murine somatic and purified germ cell subpopulations will be analyzed to correlate methylation with gene expression. After identifying genomic 5' fragments containing the promoter, capsite and first exon, subfragments will be tested functionally for tissue-specific expression and inducibility of the promoters by subcloning into suitable vectors like pSVOCat or pRSVCat. The plasmids will be introduced into tissue culture cells (CV-1) by transfection using the calcium phosphate DNA precipitation method. CAT activity can be easily assayed and the relative strength of promoter elements determined. Cis controlling elements such as enhancers, activators and repressors will be characterized by introducing 5' upstream deletion mutants into HeLa cells and mouse and human teratocarcinoma cells and assaying for appropriate expression. Sequences involved in binding trans acting factors will be identified by DNase I and Exo III footprinting experiments. Isolation and purification of the binding factors will he attempted by sequential affinity chromatography. Transgenic mice will be used as an in vivo system to test for Ldh-c gene regulation and to verify sequences that control regulation. These objectives are important to obtain an understanding of specific gene expression during spermatogenesis, and thereby impinge on questions concerning specific production from the standpoint of infertility as well as fertility control.